CN106905309B - A kind of very high selectivity and the amphiphilic fat drips fluorescence probe of tool and its application - Google Patents
A kind of very high selectivity and the amphiphilic fat drips fluorescence probe of tool and its application Download PDFInfo
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Abstract
The invention discloses a kind of very high selectivity and the amphiphilic fat drips fluorescence probes of tool, it is characterized by: the probe chemical name is 1- (3 '-(7 '-nitro benzofuraxan -4 ' -) aminopropyl) -2,3,3- tri-methyl indole bromides, abbreviation NII；Shown in its chemical general formula such as formula (I).The invention also discloses the probes to mark or show the application in fat drips form and distribution in living cells or tissue in specific manner.Experiment confirms that probe of the invention is a kind of completely new probe, has applied widely, and single, double photon good light stability, dyeing kinetics are fast, and cytotoxicity is low, and the characteristics of fat drips can exclusively be imaged in competent cell, has a extensive future.
Description
Technical field
The present invention relates to a kind of fat drips fluorescence probe and its applications more particularly to a kind of with the amphipathic of very high selectivity
Fat drips probe and its in specific manner mark or show living cells or tissue in fat drips form and distribution in application.
Background technique
Fat drips have always been considered as being to store the simple of neutral lipid (triglycerides and cholesteryl ester) in cell for a long time
Inert sphere.But recent studies have shown that fat drips are not static, but intracellular highly dynamic organelle, extensively
It is present in eukaryocyte and prokaryotic cell generally.They play important role in cell, participate in a variety of in cell
Activity, for example the metabolism of lipid within endothelial cells is adjusted, cell inner equilibrium is maintained, and interact with intracellular various kinds of cell device.And
And the exception of fat drips can lead to a variety of diseases, such as obesity, diabetes, artery congee hardening etc..Therefore in order to further investigate fat drips
Influence to fat drips of biological role and various physiological activities, the various sides including mass spectral analysis, electron microscope etc.
Method is used.In various methods, fluorescence probe can visualize to real-time in-situ fat drips, thus its become research fat drips and
The strong means of its correlated activation.
FOR ALL WE KNOW, fat drips have a big anhydrous neutral core, so according to " similar compatibility " principle, Yi Xieqin
The probe of lipid is developed and is used to that fat drips are imaged.Wherein wide application there are two commercialized fat drips probe:
Nile Red and BODIPY.Nile Red is a lipophilic probe, although it can preferentially be gathered in fat drips, it
Also to intracellular most structure dyeings, and faint fluorescence also issues in water, therefore cause great background
Noise causes selectivity very poor.In order to improve selectivity, the lipophilic probe BODIPY of another kind and other lipophilic rouge
Drop probe is also used to imaging fat drips in succession.Compared to Nile Red, their selectivity makes moderate progress, but is not still able to satisfy
Research Requirements hinder the further research of fat drips.It can be seen that merely designing lipophilicity dependent on the lipophilicity of fat drips
The difficult to realize purpose that fat drips are imaged with high selectivity of fat drips probe.Therefore, urgently need a kind of new design method with
Highly selective fat drips probe is obtained, fat drips can be targeted in specific manner, realization without background noise is imaged.
Summary of the invention
In view of the deficiencies of the prior art, the problem to be solved in the present invention is to provide a kind of with the amphipathic of very high selectivity
Fat drips fluorescence probe and its in specific manner mark or show living cells or tissue in fat drips form and distribution in application,
To realize the fat drips in for no background noise imaging cells and tissue.
Very high selectivity of the present invention and the amphiphilic fat drips fluorescence probe of tool, it is characterised in that: the probe chemical name
Referred to as 1- (3 '-(7 '-nitro benzofuraxan -4 ' -) aminopropyl) -2,3,3- tri-methyl indole bromides, abbreviation NII；Its chemistry is logical
Shown in formula such as formula (I):
The very high selectivity of the present invention and Summarization for Preparation Methods of the amphiphilic fat drips fluorescence probe (NII) of tool is as follows:
4- (3 '-the third amino of bromine) -7- benzofuraxan (compound 1) is first synthesized, then by itself and 2,3,3- tri-methyl indoles are mixed
It closes, using ethyl alcohol as solvent, is heated to reflux obtained 1- (3 '-(7 '-nitro benzofuraxan -4 ' -) aminopropyl) -2,3,3- trimethyls
Indoles bromide (NII).
Reaction equation prepared by above-mentioned very high selectivity and the amphiphilic fat drips fluorescence probe (NII) of tool is as follows:
Current report is it has been shown that the neutral core inside fat drips is wrapped by an amphiphilic Lipid monolayer
Quilt, in order to effectively store fat drips while maintain the stability of fat drips structure, the water inside fat drips is discharged, therefore actually rouge
Drop forms a unique amphiphilic structure, it includes a polar head and anhydrous inside.Recognized based on such
Know, the present invention predicts that amphiphilic molecule can target to very high selectivity fat drips, and has successfully filtered out one based on amphiphilic
The small organic molecule probe of property.This amphiphilic probe is respectively by a strong lipophilic fluorogen NBD and a cation
Partially (indoles salt) forms.Herein, we have selected the NBD for following ICT (Intramolecular electron transfer) mechanism as glimmering
Light parent, on the one hand it has strong lipophilicity, can have strong binding force with the neutral core of fat drips, in combination with cationic portion
Divide the strong electrostatic force between the polar head of fat drips, may be implemented to target fat drips in specific manner, to be conducive to nothing
Background noise it is imaged.On the other hand, the luminosity of the fluorogen by environment polarity effect, i.e., in low polar environment it
Fluorescence intensity be considerably higher than it in highly polar environment, in this way when lipophilic fluorogen targeting fat drips it is low it is polar in
Behind portion, strong fluorescence can be issued, realizes the effect of fluorescent switch, and then be imaged with being also beneficial to no background noise.Therefore, two
The dual-target strategy of parent's property probe can target fat drips in specific manner, realize the imaging without background noise.
Very high selectivity of the present invention and the amphiphilic fat drips fluorescence probe of tool are lived thin in label in specific manner or display
Born of the same parents or the fat drips form in tissue and the application in distribution.
Wherein: the preferred immortalized cells of the living cells or normal cell, the preferred Muscle Tissue of the tissue or liver
Dirty tissue.
Further, the immortalized cells are preferably HeLa cell or PC-3 cell；The normal cell is preferably
MSC cell.
Fat drips are imaged amphiphilic fluorescence provided by the invention with can be used for no background noise with very high selectivity
Probe has two photon imaging ability simultaneously.
Experimental result confirms that amphiphilic fat drips probe of the present invention has the selectivity of superelevation, being capable of specificity
Ground targets the fat drips in the even more complicated tissue of cell, and imaging effect is much better than commercialized fat drips probe Nile
red.Simultaneously as introducing cationic salt moiety non-conjugatedly on the basis of NBD will not influence the luminosity of NBD, this
So that amphiphilic probe NII maintains the photoluminescent property of NBD, i.e. ICT property, fluorescence is influenced by environment is polar, with environment
Polar increase, fluorescence intensity reduces, while Fluorescent peal red shift.Therefore when NII targeting has in low polar fat drips
When portion, fluorescence intensity will be far longer than its fluorescence intensity in water, so as to realize fluorescent switch effect.Meanwhile this
, can be without further washing after the high s/n ratio of sample and low background noise are but also probe dyed, it can be in microscope
It is lower directly to observe.NII has suitable two-photon performance simultaneously, can be used for two photon imaging.Under Two Photon Fluorescence, cell
It can be clearly visible with the fat drips in tissue.In addition, the photostability of NII probe is fabulous, hence it is evident that it is same to be better than Nile Red.
When its dyeing kinetics be exceedingly fast (2 minutes), cytotoxicity is very low, and can be compatible with other probes.Therefore probe NII can be at
For research fat drips and its strong tool of correlated activation, it is often more important that set using double targetings of amphipathic molecule
Meter thinking can provide a general guidance for the design of probe of the following film property organelle including fat drips.
Highly selective amphiphilic fat drips probe NII provided by the invention is that report for the first time amphiphilic can be used for nothing
The fluorescence probe of fat drips is imaged to background noise.Compared with other lipophilic fat drips fluorescence probes similar in its function, this hair
The bright probe NII has the selectivity of superelevation, without background noise imaging, two-photon performance, fabulous photostability, quickly dye
Color ability, it is disposable wash, the features such as cytotoxicity is low.
To sum up, probe of the invention is a kind of completely new probe, has applied widely, single, double photon good light stability,
Dyeing kinetics are fast, and cytotoxicity is low, and the characteristics of fat drips can exclusively be imaged in competent cell, have a extensive future.
Detailed description of the invention
Fig. 1: the confocal fluorescent of HeLa cell is dyed jointly with Hoechst 33342 and NII (A) or Nile red (B)
Photo.
Wherein: 1) figure is the differential interference photo of light field laser scanning；2) figure is Hoechst 33342 in 405nm laser
The fluorescence photo obtained under irradiation；3) figure is the fluorescence photo that NII or Nile red is obtained under 473nm laser irradiation；4) figure
For stacking chart 1), 2), 3)., 6), 5) 7) it is respectively enlarged drawing in corresponding yellow frame.Scale 4)=20 μm；Scale 5)
=1 μm.
Fig. 2: with Hoechst 33342 and NII (A) or Nile red (B), HeLa of the dyeing Jing Guo different disposal is thin jointly
The confocal fluorescent photo of born of the same parents.
Different disposal: 1) -4) being to fix cell, 5) -8) and it is removed into the cell by dimethylbenzene again for the cell after fixing
Fat drips.
Wherein: 1), 5) figure is the differential interference photo of light field laser scanning；2), 6) figure be Hoechst 33342 exist
The fluorescence photo obtained under 405nm laser irradiation；3), 7) figure be NII, Nile red under 473nm laser irradiation obtain
Fluorescence photo；4), 8) figure be stacking chart 2), 3).Scale=20 μm.
Fig. 3: with NII dyeing by the confocal fluorescent photo of the HeLa cell of oleic acid processing different time.
(A): 2h；(B):4h；(C):6h.
Wherein: 1) figure is the fluorescence photo that NII is obtained under 473nm laser irradiation；2) figure is light field laser scanning
Differential interference photo；3) figure is stacking chart 1), 2).Scale=20 μm.
Fig. 4: (A) figure: with the two-photon fluorescence photo of NII dyeing HeLa cell, scale=20 μm；
(B) figure: the quantization figure of the single photon photostability of the mono-/bis-photon photostability and Nile red of NII；
(C) figure: with the survival rate of NII processing HeLa cell different time (2h, 10h, for 24 hours) cell afterwards.
Fig. 5: with NII dyeing Muscle Tissue (cross section and longitudal section) and liver organization after in 840nm laser spoke
The two-photon fluorescence picture of the different depth obtained under taking.Scale=20 μm.
Specific embodiment
Embodiment 1:
The synthesis of 4- (3 '-the third amino of bromine) -7- benzofuraxan (1)
In flask, in methyl alcohol by the dissolution of 4- chloro- 7- nitrobenzofurazan (2g, 10mmol), stir 15 minutes, then plus
Enter 3- bromine propylamine (2.19g, 10mmol), reacts 8 hours at room temperature.It is extracted with dichloromethane, washes after reaction.With nothing
Aqueous sodium persulfate is dried.Column layer analysis finally, which is carried out, with the mixture of petroleum ether and ethyl acetate obtains final product.
1H NMR (300MHz, DMSO-d6): δ (ppm) 9.54 (t, J=5.40Hz, 1H), 8.54 (d, J=9.00Hz,
1H), 6.45 (d, J=8.70Hz, 1H), 3.64 (m, 4H), 2.23 (m, 2H).
The synthesis of 1- (3 '-(7 '-nitro benzofuraxan -4 ' -) aminopropyl) -2,3,3- tri-methyl indole bromide (NII)
The compound (1) (0.3g, 1mmol) of synthesis and 2,3,3- tri-methyl indoles (240 Μ l, 1.5mmol) are mixed,
Using ethyl alcohol as solvent, it is heated to reflux.After 24 hours reaction terminate, be cooled to room temperature, sediment with washed with ether three times, then again
It is rinsed with water three times, obtains final product.
1H NMR(300MHz,DMSO-d6): δ (ppm) 9.48 (s, 1H), 8.57 (d, J=9.00Hz, 1H), 7.99 (m,
1H), 7.81 (m, 1H), 7.58 (m, 2H), 6.51 (d, J=9.00Hz, 1H)13C NMR(400MHz,DMSO-d6),δ (ppm)
=197.62,144.84,142.30,141.59,138.28,129.83,129.28,123.94,115.81,100.08,
54.72, 46.12,40.68,40.47,40.26,40.05,39.84,39.63,39.39,22.52,14.61.HRMS(m/z):
[M]+ calculated for C20H22N5O3,380.43；found,380.18.
Embodiment 2: the culture of the cancer cell HeLa of immortalization
HeLa cell strain is the 5%CO at 37 DEG C2CO2It is cultivated in incubator.HeLa cell strain is including 10%
Adhere-wall culture in fetal calf serum and 1% dual anti-H-DMEM culture solution.
Equal cells grow into logarithmic phase, contact pin culture: 1. coverslip being impregnated to 30min in dehydrated alcohol, alcolhol burner dries
It is put into disposable 35mm culture dish after dry；2. the cell in 100mL cell bottle is washed three times with PBS, with 0.25% pancreas of 1mL
Enzymic digestion 3-5 minutes, culture medium is carefully poured out, a small amount of fresh culture piping and druming is added uniformly, after cell count, it is suitable to leave
The cell of density, culture medium is added to required volume, and (control final concentration of cells is 1 × 105), it is seeded to the training for including coverslip
It supports in ware, is put into CO2It is cultivated in incubator, grows cell climbing sheet.
The confocal fluorescent microscope experiment of embodiment 3:NII and Nile red the dyeing intracellular fat drips of HeLa
The cell climbing sheet connected is first used into (5 μM) the incubation 30min of nucleus dyestuff Hoechst 33342, washs 2 with PBS
Time, then 20min is incubated for 4 μM of NII stain incubation 2min or 5 μM of Nile red at room temperature.Cell climbing sheet is taken out, carefully
Intracellular growth faces lower cover on glass slide, is observed under confocal fluorescent microscopic, it is found that intracellular fat drips are clear by NII
It colours, is spherical clearly, be mainly distributed in cytoplasm (region not coloured by Hoechst).However, Nile red in addition to
It dyes except dot, has also dyed intracellular most table structure.Therefore, amphipathic probe NII of the present invention is with superelevation
The fat drips probe of selectivity, can provide the fluorescence photo without background noise of fat drips.
The result is shown in Figure 1.Dye the copolymerization of HeLa cell jointly with Hoechst 33342 and NII (A) or Nile red (B)
Burnt fluorescence photo.Wherein 1) figure be light field laser scanning differential interference photo；2) figure is that Hoechst 33342 swashs in 405nm
The fluorescence photo obtained under light irradiation；3) figure is the fluorescence photo that NII or Nile red is obtained under 473nm laser irradiation；4)
Figure is stacking chart 1), 2), 3)., 6), 5) 7) it is respectively enlarged drawing in corresponding yellow frame.Scale 4)=20 μm；Scale
=1 μm 5).
Embodiment 4: the proof experiment for proving the very high selectivity of amphipathic fat drips probe NII by removing fat drips method
Dimethylbenzene can be used to remove the fat drips in fixed cell, therefore can be used to further prove NII to the superelevation of fat drips
Selectivity.Control group: cell is first fixed with paraformaldehyde, and cell has been fixed after 30 minutes, then sucks paraformaldehyde, is added
The PBS of 1ML, then with (5 μM) the incubation 30min of nucleus dyestuff Hoechst 33342, washed 2 times with PBS, then with 4 μM
NII stain incubation 2min or 5 μM of Nile red are incubated for 20min.Experimental group: cell is first fixed with paraformaldehyde, thin after 30 minutes
Born of the same parents have been fixed, and then suck paraformaldehyde, later with the alcohol of various concentration gradient (70%, 80%, 90%, 95% He
100%) it successively handles cell each 5 minutes, then handles cell twice with dimethylbenzene, each 5 minutes, later again with anti-concentration gradient
Alcohol successively handle each 5 minutes.Cell finally is washed twice with PBS, and fat drips intracellular at this time are completely removed.Then it uses
(5 μM) the incubation 30min of nucleus dyestuff Hoechst 33342, are washed 2 times with PBS, finally with 4 μM of NII stain incubation 2min
Or 5 μM of Nile red are incubated for 20min.Cell climbing sheet in two groups of experiments is taken out, cell growth is covered in glass slide down
On, NII, the signal of Nile red and Hoechst 33342 are collected respectively in Laser Scanning Confocal Microscope.
It has been observed that intracellular fat drips can clearly be coloured by NII, but fat drips are removed it before removal fat drips
Afterwards, the fluorescence from NII is not detected.In contrast, after removing fat drips, other intracellular environment still can be by Nile red
Coloring.Therefore should the experiment proves that amphipathic probe NII is merely able to exclusively dye fat drips, and be unable in staining cell other
Environment.
As a result such as Fig. 2.With Hoechst 33342 and NII (A) or Nile red (B), different disposal is passed through in dyeing jointly
The confocal fluorescent photo of HeLa cell.Different disposal: 1) being -4) fixed cell, 5) -8) and pass through again for the cell after fixing
Dimethylbenzene removes intracellular fat drips.Wherein 1), 5) figure is the differential interference photo of light field laser scanning；2), 6) figure be
The fluorescence photo that Hoechst 33342 is obtained under 405nm laser irradiation；3), 7) figure be NII, Nile red in 473nm
The fluorescence photo obtained under laser irradiation；4), 8) figure be stacking chart 2), 3).Scale=20 μm.
Embodiment 5: the very high selectivity of amphipathic fat drips probe NII is proved again by handling HeLa cell with oleic acid
Proof experiment
Because oleic acid can be gathered in fat drips as a kind of neutral phospholipid, to can induce the formation of fat drips, make fat drips
Increase.Therefore we handle HeLa cell with oleic acid further to prove the very high selectivity of amphipathic fat drips probe NII.It will connect
Good cell climbing sheet handles different time (2h, 4h, 6h) with oleic acid respectively, is then sucked out, is washed 2 times with PBS, later with 4 μ
M NII stain incubation 2min.Cell climbing sheet is taken out, cell growth is covered on glass slide down, it is micro- in confocal fluorescent
Mirror is observed, growth of the discovery with the processing time, intracellular fat drips increase, and the green fluorescence from NII also significantly increases
By force.This shows that NII also can newly generated fat drips in staining cell well.
As a result see Fig. 3.With NII dyeing by the confocal fluorescent photo of the HeLa cell of oleic acid processing different time.
(A): 2h；(B):4h；(C): 6h. wherein 1) figure is the fluorescence photo that NII is obtained under 473nm laser irradiation；2) figure be for
The differential interference photo of light field laser scanning；3) figure is stacking chart 1), 2).Scale=20 μm.
Embodiment 6:NII dyes the two-photon photo of HeLa cell, mono-/bis-photon photostability and its cell toxicity test
Inoculated cell climbing sheet is dyed with 4 μM of NII, is incubated for 2min at room temperature.Then cell climbing sheet is taken
Out, cell growth is covered on glass slide down, directly in Two Photon Fluorescence (excitation wavelength: 840nm, average femtosecond pulse
Power is 3mW) under observe cell.The result shows that NII have two photon imaging ability, can under two-photon excitation clearly at
As the fat drips in cell.Meanwhile its mono-/bis-photon photostability and the single photon photostability of Nile red are measured, as a result table
Bright NII has fabulous photostability, hence it is evident that is higher than Nile red.With the cytotoxicity of CCK8 measurement NII, the results showed that NII pairs
The toxicity of cell is minimum.
As a result see Fig. 4.(A) with the two-photon fluorescence photo of NII dyeing HeLa cell, scale=20 μm；(B) NII
The quantization figure of mono-/bis-photon photostability and the single photon photostability of Nile red；(C) different with NII processing HeLa cell
The survival rate of time (2h, 10h, for 24 hours) cell afterwards.
The two-photon microscope experiment of embodiment 7:NII dyeing Muscle Tissue and liver organization
The musculature taken out out of Mice Body and liver organization are cultivated respectively small in the culture equipped with cell culture fluid
In ware, then dyed with 10 μM of NII, after being incubated for 2 minutes at room temperature directly under Two Photon Fluorescence (excitation wavelength:
It is observed under 840nm).
The results showed that NII can clearly in visualization organization fat drips form and distribution, and imaging depth is reachable
82μm。
As a result see Fig. 5.With after NII dyeing Muscle Tissue cross section, longitudal section and liver organization in 840nm laser spoke
The two-photon fluorescence picture of the different depth obtained under taking.
Claims (1)
1. a kind of very high selectivity and the amphiphilic fat drips fluorescence probe of tool, it is characterised in that: the probe chemical name is 1-
(3 '-(7 '-nitro benzofuraxan -4 ' -) aminopropyl) -2,3,3- tri-methyl indole bromides, abbreviation NII；Its chemical general formula such as formula
(I) shown in:
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| CN107814796B (en) * | 2017-10-16 | 2020-10-02 | 湖南大学 | Environment-sensitive dye based on benzofurazan and preparation method and application thereof |
| CN108148572B (en) * | 2018-01-12 | 2019-04-16 | 济南大学 | A kind of fat drips fluorescence probe and its synthetic method and application |
| CN108530523B (en) * | 2018-03-13 | 2020-09-15 | 上海交通大学 | Application of Arabidopsis thaliana Sec14p-like gene in fluorescent labeling of plant cell lipid droplets |
| CN108440475B (en) * | 2018-03-16 | 2020-04-07 | 济南大学 | Ratio type fluorescent probe for distinguishing lipid droplets with different polarities and preparation method and application thereof |
| CN108822019B (en) * | 2018-08-21 | 2019-08-13 | 济南大学 | Polar fluorescence probe of a kind of detection fat drips and its preparation method and application |
| CN110156713B (en) * | 2019-05-14 | 2021-07-30 | 济南大学 | Fluorescent probe for detecting lipid droplets and preparation method and application thereof |
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| CN114605456B (en) * | 2022-03-29 | 2023-06-02 | 山东大学深圳研究院 | Donor-acceptor naphthalene salicylaldehyde imine boron difluoride compound and application thereof in lipid drop super-resolution imaging |
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| CN104877665A (en) * | 2013-12-19 | 2015-09-02 | 香港科技大学深圳研究院 | Luminescent material having aggregation-induced emission, method of making and application thereof |
| CN105541660A (en) * | 2016-01-15 | 2016-05-04 | 华南理工大学 | Arylsalicylaldehyde-diphenyl-azine hydrazine compound as well as preparation and application |
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